Antioxidant, Antiapoptotic, and Anti-Inflammatory Effects of Hesperetin in a Mouse Model of Lipopolysaccharide-Induced Acute Kidney Injury

Sepsis is a severe inflammatory condition that can cause organ dysfunction, including acute kidney injury (AKI). Hesperetin is a flavonoid aglycone that has potent antioxidant and anti-inflammatory properties. However, the effect of hesperetin on septic AKI has not yet been fully investigated. This study examined whether hesperetin has a renoprotective effect on lipopolysaccharide (LPS)-induced septic AKI. Hesperetin treatment ameliorated histological abnormalities and renal dysfunction in LPS-injected mice. Mechanistically, hesperetin attenuated LPS-induced oxidative stress, as evidenced by the suppression of lipid and DNA oxidation. This beneficial effect of hesperetin was accompanied by downregulation of the pro-oxidant NADPH oxidase 4, restoration of glutathione levels, and activation of antioxidant enzymes. This flavonoid compound also inhibited apoptotic cell death via suppression of p53-dependent caspase-3 pathway. Furthermore, hesperetin alleviated Toll-like receptor 4-mediated cytokine production and macrophage infiltration. Our findings suggest that hesperetin ameliorates LPS-induced renal structural and functional injury through suppressing oxidative stress, apoptosis, and inflammation.


Introduction
Sepsis is a severe inflammatory condition caused by microbial pathogens, including bacteria, viruses, and fungi [1]. The incidence of sepsis is steadily increasing, adding to social and economic burdens [2]. Sepsis can cause organ dysfunction and is a principal cause of death in critically ill patients. Acute kidney injury (AKI) is a common complication in septic patients, and about 60% of septic patients develop AKI [3]. Indeed, sepsis is a leading cause of AKI in critically ill patients and accounts for 45-70% of all AKI cases [3]. It has also well known that AKI is associated with an increased risk of developing chronic kidney disease [4]. Current therapies for patients with septic AKI include antibiotics, fluid therapy, and vasopressors, but there is no specific treatment for the disease [3]. Therefore, the development of effective and specific therapeutic strategies for septic AKI is urgently needed.
Among the microbial pathogens, Gram-negative bacteria (GNB) are known to be frequently involved in septic AKI [5]. Lipopolysaccharide (LPS) is the major structural component of the membrane of GNB [6]. LPS is recognized by Toll-like receptor 4 (TLR4), which is expressed on renal cells, such as tubular epithelial cells, as well as immune cells, such as macrophages [7]. Upon activation, TLR4 mediates its function in the inflammatory response by recruiting the cytoplasmic protein, myeloid differentiation primary response 88 (MyD88) [7]. During Gram-negative bacteremia, the stimulation of TLR4 by LPS in immune cells leads to the overproduction of proinflammatory cytokines and reactive oxygen species (ROS) as well as the infiltration of immune cells into the damaged organ [8,9]. Exposure of LPS to tubular epithelial cells also induces ROS production and apoptotic cell death [10,11]. Eventually, these changes result in structural and functional renal injury. As such, because of the essential role of LPS in the pathophysiology of septic AKI, rodent models of LPS injection are widely used to study mechanisms of sepsis-induced organ dysfunction and discover new therapies [12].

Discussion
Hesperetin is a flavonoid mainly found in citrus fruits and has been known to possess antioxidant and anti-inflammatory activities [13,14]. Accumulating evidence suggests that this compound exerts beneficial effects on a variety of inflammatory diseases,

Discussion
Hesperetin is a flavonoid mainly found in citrus fruits and has been known to possess antioxidant and anti-inflammatory activities [13,14]. Accumulating evidence suggests that this compound exerts beneficial effects on a variety of inflammatory diseases, including obstructive kidney disease and diabetic nephropathy [15][16][17][18][19][20][21][22][23]. In this study, hesperetin alleviated structural and functional renal injury in LPS-injected mice, as evidenced by the amelioration of histological tubular damage, attenuation of the brush border loss, downregulation of the tubular injury marker, and a reduction in serum creatinine and BUN levels. AKI can result from many types of causes, including hypoperfusion, sepsis, and exposure to nephrotoxins [36]. Sepsis has a complex and unique pathophysiology, making septic AKI a syndrome distinct from other types of AKI [8]. Similar to our findings, recent studies have shown that hesperetin attenuates cisplatin-induced histological abnormalities and renal dysfunction in rodents [24,25]. Cisplatin is a widely used chemotherapeutic agent with a high potential for nephrotoxicity [37,38]. Taken together, these results suggest that the renoprotective effect of hesperetin is not limited to a specific type and can act on AKI caused by various insults, including nephrotoxins and sepsis.
Oxidative stress plays a critical role in the pathophysiology of septic AKI [8.9]. Indeed, LPS injection induces ROS production in the kidney [39][40][41]. Because hesperetin is known to exert potent antioxidant activity [13,14], we assessed the effect of hesperetin on oxidative stress. In this study, hesperetin reduced the amounts of lipid (4-HNE and MDA) and DNA (8-OHdG) oxidation products in LPS-injected mice, indicating that hesperetin inhibits LPS-induced oxidative stress. This effect of hesperetin was accompanied by NOX4 downregulation. NOX4 is an enzyme that plays a critical role in ROS production and oxidative stress in renal diseases [42]. Numerous studies have reported the upregulation of NOX4 in AKI models caused by various insults including LPS [43,44] and cisplatin [45,46]. Yoo et al. showed that NOX4-mediated ROS production induces histological abnormalities and renal failure in LPS-induced AKI [47]. Therefore, the downregulation of NOX4 induced by hesperetin may be critically involved in its inhibitory action on oxidative stress. In addition, antioxidant enzymes play an important role in defense against LPS-induced AKI [48]. In this study, the primary endogenous antioxidant enzymes, catalase and MnSOD, were downregulated by LPS injection, but hesperetin significantly reversed the expression of these enzymes. Activities of catalase and SOD were also increased by hesperetin. GHS is an endogenous antioxidant that exerts a protective effect against oxidative damage [48]. Hesperetin attenuated LPS-induced depletion of GSH in the kidney. Similar to our findings, hesperetin inhibited antioxidant enzymes to suppress oxidative stress and inflammatory responses in various disease models [49][50][51]. Altogether, these results suggest that hesperetin inhibits LPS-induced oxidative stress via modulation of pro-oxidant and antioxidant enzymes.
Tubular cell apoptosis is an important pathological mechanism of septic AKI [8,9]. Previous studies have reported that a higher number of apoptotic cells were observed in LPS-injected mice [10,11]. In this study, we performed TUNEL assay to detect apoptotic cells. Hesperetin significantly inhibited apoptosis in LPS-injected mice. Cleavage of caspase-3 and PARP-1 was also attenuated by hesperetin. In addition, hesperetin reduced the protein levels of p53 and the mRNA expression of its transcriptional targets, PUMA and Bax. These results suggest that hesperetin attenuates LPS-induced apoptosis via inhibition of p53-dependent caspase-3 pathway. Consistent with these results, hesperetin inhibited apoptotic cell death in animal models of sorafenib-induced cardiotoxicity [52], doxorubicininduced pulmonary toxicity [53], and acetaminophen-induced hepatotoxicity [54].
Cytokine overproduction and immune cell infiltration are hallmarks of septic AKI [8,9]. Rodents injected with LPS have been shown to exhibit increased serum and renal levels of cytokines and marked infiltration of immune cells into the injured kidney [55,56]. LPS triggers an inflammatory response by stimulating TLR4 and recruiting MyD88 in immune cells and renal tubular epithelial cells [7][8][9]. Eventually, TLR4-MyD88 pathway activates the transcription factor NF-κB. Accumulating evidence points to the essential role of the TLR4 pathway in the pathogenesis of septic AKI [57]. In this study, we found that hesperetin suppressed cytokine overproduction and macrophage infiltration in LPS-injected mice. LPS injection activated the TLR4-MyD88-NF-κB signaling pathway, but this pathway was significantly inhibited by hesperetin. Wang et al. reported that hesperetin alleviates LPS-induced acute lung injury in mice via the inhibition of the TLR4-MyD88-NF-κB pathway [20]. Zhang et al. also reported that hesperetin inhibited the polarization of microglia and exerted a protective effect in a mouse model of ischemic stroke by suppressing the TLR4-NF-κB pathway [58]. Overall, these results suggest that the beneficial action of hesperetin on LPS-induced cytokine production and macrophage infiltration may be mainly due to the suppression of the TLR4-MyD88-NF-κB cascade.
Our study has several limitations. First, the dose-dependent effects of hesperetin were not evaluated. Characterizing the dose-dependent effects of a compound can provide important insights into its efficacy and application. Second, the experimental design did not include a vehicle control group. Compared to an untreated control, a vehicle control can determine whether the vehicle alone causes any effects.
In conclusion, our data demonstrated that hesperetin exerts a protective effect against LPS-induced structural and functional renal injury. Hesperetin inhibited LPS-induced oxidative stress via modulation of pro-oxidant and antioxidant enzymes. In addition, p53-mediated apoptosis was alleviated by hesperetin. This compound also attenuated TLR4-dependent cytokine production and macrophage infiltration. Although future studies will be needed to further elucidate the mechanism of action of hesperetin, our findings suggest that it could be a potential therapeutic option for septic AKI.

Animal Experiments
Eight-week-old male C57BL/6N mice were acquired from HyoSung Science (Daegu, Korea) and were caged at a temperature of 22 ± 2 • C and a humidity of 55 ± 5% under a 12/12 h light-dark cycle. The mice were randomly divided into three groups: a control group (n = 8), LPS group (n = 8), and LPS+Hes group (n = 8). Septic AKI was induced using a single intraperitoneal injection of LPS (10 mg/kg; dissolved in normal saline). Hesperetin (50 mg/kg) or vehicle (DMSO; 1 mL/kg) was administered intraperitoneally 1 h after LPS injection. LPS and hesperetin were obtained from Sigma-Aldrich (St. Louis, MO, USA). The dose of hesperetin was determined based on a previous study investigating its renoprotective effect in mice [24]. Mice were sacrificed at 24 h after LPS injection. Blood samples were collected using cardiac puncture. The right kidney was fixed in 10% formalin for histological analysis, and the left kidney was frozen in liquid nitrogen for protein and mRNA analysis. Animal experiments were approved by the Institutional Animal Care and Use Committee of the Daegu Catholic University Medical Center (DCIAFCR-221007-27-Y).

TUNEL Assay
Apoptotic cells were detected using a TUNEL assay kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's protocol. Briefly, kidney sections were deparaffinized, rehydrated, and permeabilized. After being washed, the sections were incubated in the TUNEL reaction mixture. Nuclei were stained with DAPI. Positive cells were counted in 10 random cortical fields (×600) per sample.

Biochemial Analyses in Blood and Kidney
Serum creatinine and BUN levels were determined using a biochemical autoanalyzer (Hitachi, Osaka, Japan). Renal MDA and 8-OHdG levels were analyzed using the MDA assay kit (Sigma-Aldrich, St. Louis, MO, USA) and the 8-OHdG ELISA kit (Abcam, Cambridge, MA, USA), respectively. Renal GSH and GSSG levels were analyzed using the GSH detection kit (Enzo Life Sciences, Farmingdale, NY, USA). Catalase and SOD activities were determined using colorimetric activity kits (Invitrogen, Carlsbad, CA, USA). Serum TNF-α and IL-6 levels were determined using ELISA kits (R&D Systems, Minneapolis, MN, USA). All assays were performed according to the manufacturers' protocols.

qRT-PCR
Total RNA was extracted with TRIzol reagent. cDNA was synthesized from extracted RNA using a reverse transcription kit (TaKaRa, Tokyo, Japan). The transcriptional level was analyzed using qRT-PCR with SYBR Premix Ex Taq II (TaKaRa, Tokyo, Japan) and primers (Table 1) in the Thermal Cycler Dice Real Time System III (TaKaRa, Tokyo, Japan). GAPDH was used as the reference gene, and relative levels of mRNA were calculated using the 2 −∆∆CT method.

Statistical Analysis
Data are presented as the mean ± SEM. Statistical significance was analyzed with one-way analysis of variance (ANOVA) and Bonferroni's tests. A p-value less than 0.05 was considered significant.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data supporting the findings of this study are available within the article.